Proton alleviation of growth inhibition by toxic metals (Al, La, Cu) in rhizobia

Free-living rhizobia are sensitive to soils and artificial media that are acidic. Both excessive H+ and Al released from acid-soluble minerals appear to be toxic. The complex, heterotrophic nutrient requirements of rhizobia and the joint occurrence of Al3+ and hydroxo-, sulphato-, phosphato-, fluoro-, and other Al species have prevented a precise attribution of toxicity to the Al species. In the present study, a medium composed of 0.3 mM MgSO4, 2 mM CaCl2, and 10 mM sucrose (the basal medium) enabled a 1000-fold cell increase at pH 4.6 or above. Additions of 1 μM AlCl3 to the basal medium were highly intoxicating, especially at higher pH: below pH 5.0 cell numbers increased slightly; at pH 5.0 cell numbers did not change from the inoculum; at higher pH values the cell numbers declined. Similar trends were observed for La3+ and Cu2+ intoxication. Uptake of methylene blue, a positively charged dye useful as a probe of cell-surface electrical potential, was inhibited by pH reductions between pH 3.5 and 6.0. Factors that decrease cell-surface negativity (such as lower pH) reduce the intoxication by cations in plant roots, but the pH responsiveness of the rhizobia in our system was much greater than the pH responsiveness of plants. Although plant-root intoxication by mononuclear hydroxo-Al species has been discounted, rhizobia may be sensitive to those species. These results have implications for the management of rhizobia in acidic soils and for the development of resistant strains.